The effects of 3-acetyldeoxynivalenol (3-AcDON) on mitogen-induced lymphocyte proliferation and antibody production were studied in male CD-1 mice exposed to 0, 2.5, 5 or 10 ppm 3-AcDON in the diet for 35 days. Mitogen-induced lymphocyte proliferation and T-cell-independent antibody responses to dinitrophenyl-ficoll or Escherichia coli were not altered by dietary exposure to 3-AcDON. The T-cell-dependent antibody response to sheep red blood cells was increased in the group fed 10 ppm 3-AcDON. In vitro, 3-AcDON inhibited lymphocyte proliferation in a dose-dependent manner. Inhibition was observed when the toxin was present during the first 8 hr in phytohaemagglutinin-stimulated cultures and during the first 24 hr in lipopolysaccharide-stimulated cultures. This suggests that 3-AcDON blocks an early step in lymphocyte activation. This inhibition was not restored by thiol reducing agents (dithiothreitol, L-cysteine or 2-mercaptoethanol). Similarly, the addition of lymphokines, including interleukin-1 or interleukin-2, did not alter the inhibitory effects of 3-AcDON. These results suggest that the in vitro effects of 3-AcDON may not reflect its in vivo immunotoxicity. However, 3-AcDON may serve as a chemical probe for examining the activation process of lymphocyte proliferation.